Inhibitors of thromboxane formation and action

ABSTRACT

Methods and compositions employing as active ingredient hepoxilins and hepoxilin analog useful for inhibiting thromboxane formation and antagonising thrombbxane activity. Methods and compositions employing these compounds provide treatment for a number of disease conditions.

FIELD OF THE INVENTION

[0001] The present invention relates to therapeutic methods andpharmaceutical compositions employing compounds which inhibitthromboxane formation and antagonise thromboxane activity.

BACKGROUND OF THE INVENTION

[0002] The maintenance of normal vascular tone in mammals involves ahomeostatic balance between factors which promote vasodilation andfactors which promote vasoconstriction. Thromboxane A₂ (TxA₂), forexample, is a powerful vasoconstrictor (1) and also a potent mediator ofplatelet aggregation (14). Prostaglandins such as prostaglandin E₂ andprostacyclin, on the other hand, have vasodilatory and anti-plateletaggregation effects.

[0003] Many diseases involve a perturbation of this normal homeostaticbalance. For example, diabetes mellitus (5-7), hypertension (8-13),thrombosis (14-16) and septic shock (17-19) are associated with animbalance of the thromboxane: prostacyclin or thromboxane: prostaglandinE₂ ratios in favour of thromboxane. There is therefore considerableinterest in finding ways of selectively controlling thromboxaneformation, so as to block the vasoconstrictor and aggregatory componentof the prostaglandin system (thromboxane) without affecting thevasodilator and anti-aggregatory prostaglandins.

[0004] Prostaglandins, including thromboxane, are formed from a commonprecursor, PGH₂. This precursor is formed through the action ofcyclooxygenase (COX) and is transformed by specific enzymes into each ofthe prostaglandins and thromboxane. Platelets convert PGH₂ selectivelyinto thromboxane A₂ through the action of the PGH₂ metabolizing enzyme,thromboxane A₂ synthase. In other tissues and cells, PGH₂ is convertedinto other prostaglandin types, e.g. prostaglandin E₂ and I₂. Sincethromboxane A₂ (TxA₂) has a very short half life in the body (30 sec),stable analogs of TxA₂ which mimic its actions have been explored; twosuch analogs are U44609 (20, 21) and U46619 (22). These analogs causeactions similar to TxA₂, i.e. they cause platelet shape change andaggregation, as well as contraction of smooth muscle. These TxA₂ mimicshave also been used to develop inhibitors of TxA₂ action as they workthrough the activation of the thromboxane receptors called TP receptors.

[0005] Several thromboxane synthase inhibitors (TSI) have been reported,some based on imidazole (23). In general, the IC₅₀'s of theimidazole-based inhibitors were in the range 10⁻⁴-10⁻⁷ m (24).Undesirable pressor effects that could not be resolved from theiranti-thrombotic actions have been noted upon administration of thesedrugs, making the substituted imidazoles unsuitable for drugdevelopment.

[0006] Non-steroidal anti-inflammatory drugs such as aspirin have alsobeen used to inhibit thromboxane synthesis. Aspirin, however, hasfrequent side effects, including gastric ulcers and Reye's syndrome.Attempts have been made to avoid these problems by developing COX-2inhibitors such as Celebrex and VIOXX (42, 43). These drugs do not,however affect thromboxane formation in platelets where COX-1 is presentand therefore are not of assistance in combatting thrombosis.

[0007] The hepoxilins are biologically active metabolites of arachidonicacid formed through the 12(S)-lipoxygenase pathway and hence arestructurally unlike the prostaglandin endoperoxides (25-27). Fournatural hepoxilins have been identified, the A-type hepoxilinsconsisting of two epimers having a hydroxyl group at carbon 8 (8(S,R)-hydroxy-11 (S), 12(S)-epoxy-eicosa -5Z, 9E, 14Z-trienoic acid) andthe B-type, two epimers having a hydroxyl group at carbon 10(10(S,R)-hydroxy-11(S), 12(S)-epoxy-eicosa-5Z, 8Z,14Z-trienoic acid).Pharmacological studies have provided evidence that these compoundsraise intracellular calcium by activating calcium stores in humanneutrophils (28, 29). A hepoxilin-specific receptor responsible for thisaction has been suggested (30). The hepoxilins activate potassiumchannels in platelets (31) and in the Aplysia brain (32, 33). Inplatelets, the hepoxilins are formed endogenously in response of thecell to hypertonic volume expansion, and function to normalize cellvolume (31).

[0008] Neither the hepoxilins nor the hepoxilin analogs described hereinhave previously been reported to affect thromboxane formation andaction.

SUMMARY OF THE INVENTION

[0009] The present invention provides new methods and compositions forinhibiting thromboxane formation and antagonising thromboxane activityin mammals.

[0010] These methods can be used to treat thromboxane-mediated diseasesincluding cardiovascular diseases, diabetes mellitus, hypertension,thrombosis and septic shock, or any disorder where it is desirable toreduce thromboxane formation and/or activity.

[0011] The invention further provides a method for treating eyediseases, associated with ocular inflammation and/or increased ocularpressure, by administration of hepoxilins or hepoxilin analogs asdescribed herein

[0012] In accordance with one embodiment, the present invention providesa method for inhibiting thromboxane formation in a mammal comprisingadministering to the mammal an effective amount of a hepoxilin or of ahepoxilin analog of the formula:

[0013] wherein X is O, CH₂, S or NH;

[0014] R¹ is lower alkyl or alkene;

[0015] lower alcohol (C1 to C22), saturated or unsaturated; or—CH₂CH═CH— (CH₂)₃—COR″ wherein R″ is OH, O— lower alkyl or alkene;

[0016] R² is OH, NH₂, SH, OPO₃H, lower alkyl or alkene or O— lower alkylor alkene; and

[0017] R³ is lower alkyl or alkene or —CH₂—CH═CH—(CH₂)₄—R′″ wherein R′″is CH₃, CH₂OH, CH₂—O— lower alkyl or alkene, phenyl or substitutedphenyl

[0018] wherein X, R¹, R² and R³ are as defined for formula I and R⁴ islower alkyl or alkene;

[0019] lower alcohol (C1 to C22), saturated or unsaturated; or

—CH═CH—CH₂—CH═CH—(CH₂)₃—COR″

[0020] wherein R″=OH or O— lower alkyl or alkene, or of a derivativethereof.

[0021] In accordance with a further embodiment, the present inventionprovides a method for inhibiting thromboxane formation in a mammalcomprising administering to the mammal an effective amount of a compoundselected from the group consisting of:

[0022] (a) 8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoicacid or a derivative thereof;

[0023] (b) 8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoicacid or a derivative thereof;

[0024] (c) 10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoicacid or a derivative-thereof; and

[0025] (d) 10(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoicacid or a derivative thereof.

[0026] In accordance with a further embodiment, the present inventionprovides a method for antagonising thromboxane activity in a mammalcomprising administering to the mammal an effective amount of ahepoxilin or of a hepoxilin analog of the formula:

[0027] wherein X is O, CH₂, S or NH;

[0028] R¹ is lower alkyl or alkene;

[0029] lower alcohol (C1 to C22), saturated or unsaturated; or

[0030] —CH₂CH═CH—(CH₂)₃—COR″ wherein R″ is OH, O— lower alkyl or alkene;

[0031] R² is OH, NH₂, SH, OPO₃H, lower alkyl or alkene or O— lower alkylor alkene; and

[0032] R³ is lower alkyl or alkene or

[0033] —CH₂—CH═CH—(CH₂)₄—R′″ wherein R′″ is CH₃, CH₂OH, CH₂ O— loweralkyl or alkene, phenyl or substituted phenyl or

[0034] wherein X, R¹, R² and R³ are as defined for formula I and R⁴ islower alkyl or alkene;

[0035] lower alcohol (C1 to C22), saturated or unsaturated; or

—CH═CH—CH₂—CH═CH—(CH₂)₃—COR″

[0036] wherein R″=OH or O— lower alkyl or alkene, or of a derivativethereof.

[0037] In accordance with a further embodiment, the present inventionprovides a method for antagonising thromboxane activity in a mammalcomprising administering to the mammal an effective amount of a compoundselected from the group consisting of:

[0038] (a) 8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoicacid or a derivative thereof;

[0039] (b) 8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoicacid or a derivative thereof;

[0040] (c) 10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoicacid or a derivative thereof; and

[0041] (d) 10(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoicacid or a derivative thereof.

[0042] In accordance with a further embodiment, the present inventionprovides a method of preventing or reducing thromboxane-mediatedplatelet aggregation in a mammal comprising administering to the mammalan effective amount of a hepoxilin or of a hepoxilin analog of theformula:

[0043] wherein X is O, CH₂, S or NH;

[0044] R¹ is lower alkyl or alkene;

[0045] lower alcohol (C1 to C22), saturated or unsaturated; or

[0046] —CH₂CH═CH—(CH₂)₃—COR″ wherein R″ is OH, O— lower alkyl or alkene;

[0047] R² is OH, NH₂, SH, OPO₃H, lower alkyl or alkene or O— lower alkylor alkene; and

[0048] R³ is lower alkyl or alkene or

[0049] —CH₂—CH═CH—(CH₂)₄—R′″ wherein R′″ is CH₃, CH₂OH, CH₂ —O— loweralkyl or alkene, phenyl or substituted phenyl

[0050] wherein X, R¹, R² and R³ are as defined for formula I and R⁴ islower alkyl or alkene;

[0051] lower alcohol (C1 to C22), saturated or unsaturated; or

[0052] ti —CH═CH—CH₂—CH═CH—(CH₂)₃—COR″

[0053] wherein R″=OH or O— lower alkyl or alkene or a derivativethereof.

[0054] In accordance with a further embodiment, the present inventionprovides a method of preventing or reducing thromboxane-mediatedplatelet aggregation in a mammal comprising administering an effectiveamount of a compound selected from the group consisting of:

[0055] (a) 8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoicacid or a derivative thereof;

[0056] (b) 8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoicacid or a derivative thereof;

[0057] (c) 10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoicacid or a derivative thereof; and

[0058] (d) 10(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoicacid or a derivative thereof.

[0059] In accordance with a further embodiment, the present inventionprovides a method of treating a thromboxane-mediated disease in a mammalcomprising administering to the mammal an effective amount of ahepoxilin or of a hepoxilin analog of the formula:

[0060] wherein X is O, CH₂, S or NH;

[0061] R¹ is lower alkyl or alkene;

[0062] lower alcohol (C1 to C22), saturated or unsaturated; or—CH₂CH═CH—(CH₂)₃—COR″ wherein R″ is OH, O— lower alkyl or alkene;

[0063] R² is OH, NH₂, SH, OPO₃H, lower alkyl or alkene or O— lower alkylor alkene; and

[0064] R³ is lower alkyl or alkene or —CH₂—CH═CH—(CH₂)₄—R′″ wherein R′″is CH₃, CH₂OH, CH₂ O— lower alkyl or alkene, phenyl or substitutedphenyl or

[0065] wherein X, R¹, R² and R³ are as defined for formula I and R⁴ islower alkyl or alkene;

[0066] lower alcohol (C1 to C22), saturated or unsaturated; or

—CH═CH—CH₂—CH═CH—(CH₂)₃—COR″

[0067] wherein R″=OH or O— lower alkyl or alkene or a derivativethereof.

[0068] In accordance with a further embodiment, the present inventionprovides a method of treating a thromboxane-mediated disease in a mammalcomprising administering to the mammal an effective amount of a compoundselected from the group consisting of:

[0069] (a) 8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z, 10E,14Z-trienoicacid or a derivative thereof;

[0070] (b) 8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoicacid or a derivative thereof;

[0071] (c) 10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoicacid or a derivative thereof; and

[0072] (d) 10(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoicacid or a derivative thereof.

[0073] In accordance with a further embodiment, the present inventionprovides a method for treating or preventing an eye disease associatedwith ocular inflammation and/or increased intraocular pressurecomprising administering to the mammal an effective amount of ahepoxilin or of a hepoxilin analog of the formula:

[0074] wherein X is O, CH₂, S or NH;

[0075] R¹ is lower alkyl or alkene;

[0076] lower alcohol (C1 to C22), saturated or unsaturated; or

[0077] —CH₂CH═CH—(CH₂)₃—COR″ wherein R″ is OH, O— lower alkyl or alkene;

[0078] R² is OH, NH₂, SH, OPO₃H, lower alkyl or alkene or O— lower alkylor alkene; and

[0079] R³ is lower alkyl or alkene or

[0080] —CH₂—CH═CH—(CH₂)₄—R′″ wherein R′″ is CH₃, CH₂OH, CH₂

[0081] —O— lower alkyl or alkene, phenyl or substituted phenyl or

[0082] wherein X, R¹, R² and R³ are as defined for formula I and R⁴ islower alkyl or alkene;

[0083] lower alcohol (C1 to C22), saturated or unsaturated; or

—CH═CH—CH₂—CH═CH—(CH₂)₃—COR″

[0084] wherein R″=OH or O— lower alkyl or alkene or a derivativethereof.

[0085] In accordance with a further embodiment, the present inventionprovides a method for treating or preventing an eye disease associatedwith ocular inflammation and/or increased intraocular pressurecomprising administering to the mammal an effective amount of a compoundselected from the group consisting of:

[0086] (a) 8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoicacid or a derivative thereof;

[0087] (b) 8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoicacid or a derivative thereof;

[0088] (c) 10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoicacid or a derivative thereof; and

[0089] (d) 10(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoicacid or a derivative thereof.

[0090] In accordance with a further embodiment, the present inventionprovides a method for reducing intra-ocular pressure in a mammalcomprising administering intra-ocularly to the mammal an effectiveamount of at least one of a hepoxilin or of a hepoxilin analog of theformula:

[0091] wherein X is O, CH₂, S or NH;

[0092] R¹ is lower alkyl or alkene;

[0093] lower alcohol (C1 to C22), saturated or unsaturated; or

[0094] —CH₂CH═CH—(CH₂)₃—COR″ wherein R″ is OH, O— lower alkyl or alkene;

[0095] R² is OH, NH₂, SH, OPO₃H, lower alkyl or alkene or O— lower alkylor alkene; and

[0096] R³ is lower alkyl or alkene or

[0097] —CH₂—CH═CH—(CH₂)₄—R′″ wherein R′″ is CH₃, CH₂OH, CH₂ O— loweralkyl or alkene, phenyl or substituted phenyl or

[0098] wherein X, R¹, R² and R³ are as defined for formula I and R⁴ islower alkyl or alkene;

[0099] lower alcohol (C1 to C22), saturated or unsaturated; or

—CH═CH—CH₂—CH═CH—(CH₂)₃—COR″

[0100] wherein R″=OH or O— lower alkyl or alkene or a derivativethereof.

[0101] In accordance with a further embodiment, the present inventionprovides a method for reducing intra-ocular pressure in a mammalcomprising administering intra-ocularly to the mammal an effectiveamount of a compound selected from the group consisting of:

[0102] (a) 8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E, 14Z-trienoicacid or a derivative thereof;

[0103] (b) 8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoicacid or a derivative thereof;

[0104] (c) 10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoicacid or a derivative thereof; and

[0105] (d) 10(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoicacid or a derivative thereof.

[0106] In accordance with a further embodiment, the present inventionprovides use of a compound of the formula:

[0107] wherein X is O, CH₂, S or NH;

[0108] R¹ is lower alkyl or alkene;

[0109] lower alcohol (C1 to C22), saturated or unsaturated; or

[0110] —CH₂CH═CH—(CH₂)₃—COR″ wherein R″ is OH, O— lower alkyl or alkene;

[0111] R² is OH, NH₂, SH, OPO₃H, lower alkyl or alkene or O— lower alkylor alkene; and

[0112] R³ is lower alkyl or alkene or

[0113] —CH₂—CH═CH—(CH₂)₄—R′″ wherein R′″ is CH₃, CH₂OH, CH₂ —O— loweralkyl or alkene, phenyl or substituted phenyl

[0114] wherein X, R¹, R² and R³ are as defined for formula I and R⁴ islower alkyl or alkene;

[0115] lower alcohol (C1 to C22), saturated or unsaturated; or

—CH═CH—CH₂—CH═CH—(CH₂)₃—COR″

[0116] wherein R″=OH or O— lower alkyl or alkene, or a derivativethereof, for the preparation of a medicament for a treatment selectedfrom the group consisting of:

[0117] (a) for inhibiting thromboxane formation in a mammal;

[0118] (b) for treating or preventing eye disease associated with ocularinflammation and/or increased intraocular pressure in a mammal;

[0119] (c) for inhibiting thromboxane activity in a mammal;

[0120] (d) for preventing or reducing thromboxane-mediated plateletaggregation in a mammal;

[0121] (e) for treating a thromboxane-mediated disease in a mammal; and

[0122] (f) for reducing intra-ocular pressure in a mammal.

[0123] In accordance with a further embodiment, the present inventionprovides use of a compound selected from the group consisting of:

[0124] (a) 8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoicacid or a derivative thereof;

[0125] (b) 8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoicacid or a derivative thereof;

[0126] (c) 10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoicacid or a derivative thereof; and

[0127] (d) 10(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoicacid or a derivative thereof,

[0128] for a treatment selected from the group consisting of:

[0129] (a) for inhibiting thromboxane formation in a mammal;

[0130] (b) for treating or preventing eye disease associated with ocularinflammation and/or increased intraocular pressure in a mammal;

[0131] (c) for inhibiting thromboxane activity in a mammal;

[0132] (d) for preventing or reducing thromboxane-mediated plateletaggregation in a mammal;

[0133] (e) for treating a thromboxane-mediated disease in a mammal; and

[0134] (f) for reducing intra-ocular pressure in a mammal.

[0135] In accordance with a further embodiment, the present inventionprovides a composition for application to the eye comprising as activeingredient a compound of the formula:

[0136] wherein X is O, CH₂, S or NH;

[0137] R¹ is lower alkyl or alkene;

[0138] lower alcohol (C1 to C22), saturated or unsaturated; or

[0139] —CH₂CH═CH—(CH₂)₃—COR″ wherein R″ is OH, O— lower alkyl or alkene;

[0140] R² is OH, NH₂, SH, OPO₃H, lower alkyl or alkene or O— lower alkylor alkene; and

[0141] R³ is lower alkyl or alkene or

[0142] —CH₂—CH═CH—(CH₂)₄—R′″ wherein R′″ is CH₃, CH₂OH, CH₂ —O— loweralkyl or alkene, phenyl or substituted phenyl or

[0143] wherein X, R¹, R² and R³ are as defined for formula I and R⁴ islower alkyl or alkene;

[0144] lower alcohol (C1 to C22), saturated or unsaturated; or

—CH═CH—CH₂—CH═CH—(CH₂)₃—COR″

[0145] wherein R″=OH or O— lower alkyl or alkene, or a derivativethereof.

[0146] In accordance with a further embodiment, the present inventionprovides a composition for application to the eye comprising as activeingredient a compound selected from the group consisting of:

[0147] (a) 8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoicacid or a derivative thereof;

[0148] (b) 8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoicacid or a derivative thereof;

[0149] (c) 10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoicacid or a derivative thereof; and

[0150] (d) 10(R)hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acidor a derivative thereof.

SUMMARY OF THE DRAWINGS

[0151] Certain embodiments of the invention are described, referencebeing made to the accompanying drawings, wherein:

[0152]FIG. 1 shows inhibitory effects of four hepoxilin analogs, PBT-1to PBT-4 on the collagen-evoked aggregation of human platelets. Typicalinhibitory curves are shown. The PBT compounds (1 μg each) were added towashed platelets in a cuvette 2 min prior to the addition of collagen (2μg) to the cuvette containing 350×106 platelets in 0.5 ml volume.

[0153]FIG. 2 shows dose response curves for the anti-aggregatory effectsof the four PBT compounds shown in FIG. 1. For comparison, the effectsof the four native hepoxilins are shown. The corresponding IC50 for eachcompound is shown.

[0154]FIG. 3 shows dose response curves for the inhibitory effect of thefour PBT compounds on TxB2 formation evoked by collagen. Experimentaldetails are as shown in FIG. 1. The contents of the cuvettes, afteraggregation was complete (5 min), were extracted with ethyl acetate asdescribed in the Methods section, and the extracted compounds wereconverted into the ADAM-acetate fluorescent derivatives and TxB2 wasquantified by HPLC. The IC50 for TxB2 inhibition compares favourablywith that for inhibition of aggregation shown in FIG. 2.

[0155]FIG. 4 shows inhibition of PGH2-evoked platelet aggregation byPBT-3. PGH2 was used at 100 ng, and the amounts of PBT-3 are shown foreach inhibitory curve.

[0156]FIG. 5 shows metabolism of [3H]-PGH2 by human platelets andinhibition of formation of tritiated TxB2 by PBT-3. Tritiated PGH2 wasadmixed with unlabeled PGH2 and added to platelet suspensions in acuvette 2 min after the addition of PBT-3. Aggregation (or itsinhibition) was followed as shown in FIG. 4. The reaction was stopped attwo minutes after addition of PGH2 and the contents of the cuvette wereextracted with ethyl acetate. The distribution of radioactivity amongvarious products was assessed by TLC.

[0157]FIG. 6 shows the inhibitory effect of PBT-3 on plateletaggregation evoked by the TxA₂ mimic, U46619.

[0158]FIG. 7A shows the effect of various doses of the hepoxilin analogPBT-3 on platelet aggregation evoked by I-BOP. Values shown are typicalof three separate experiments.

[0159]FIG. 7B shows the effect of various doses of the hepoxilin analogPBT-3 on platelet aggregation evoked by the thromboxane analog U46619.

[0160]FIG. 8 shows dose response curves for the inhibition by PBT-3 ofplatelet aggregation evoked by collagen, U46619 and I-BOP. Valuesrepresent data from three separate experiments ±SD.

[0161]FIG. 9 shows dose response curves for the inhibition of binding of¹²⁵I-BOP binding to platelets by U46619, PBT-3 and I-BOP.

[0162]FIG. 10 shows the inhibitory effect of PBT-3 on plateletaggregation evoked by collagen or U46619 in the presence of ASA. Arrowsindicate the time of addition of the various substances identified bynumber.

[0163]FIG. 11 shows intra-ocular pressure (IOP) in treated (t) andcontrol (c) eyes of rats; treatments were A: 0.005% latanoprost +0.5%o-bunolol; B: 0.005% latanoprost; C: 0.005% PBT-3; D: 0.005% PBT-30; andE: 0.05% o-bunolol. Open bar on far left is mean IOP of all control(untreated) eyes (n=129).

[0164]FIG. 12 is a schematic diagram of the chemical synthesis ofgalactose amide derivatives of hepoxilin analogs.

[0165]FIG. 13 is a schematic diagram of the chemical synthesis of aprotected galactose intermediate. Reagents and conditions: i, Ac₂O, Py;ii, 90% TFA/H₂O; iii, BDMSCI, ImH, DMF; iv, 1N NaOH, MeOH—H₂O.

[0166]FIG. 14 is a schematic diagram of the chemical synthesis ofhepoxilin galactose esters. Reagents and conditions: i, RCOOH (7a,b),EDAC, DMAP, CH₂Cl₂; ii, n-Bu₄NF, Py.HCl, THF

DETAILED DESCRIPTION OF THE INVENTION

[0167] In accordance with one embodiment, the present invention providesmethods and pharmaceutical compositions for inhibiting thromboxaneformation in a mammal.

[0168] These methods and compositions employ as active ingredient anatural hepoxilin or a hepoxilin analog.

[0169] As used herein, a “hepoxilin” means a naturally occurringhepoxilin. Naturally occurring hepoxilins include A-type hepoxilinsconsisting of two epimers having a hydroxyl group at carbon 8 (8(S,R)-hydroxy-11 (S), 12(S)-epoxy-eicosa-5Z, 9E, 14Z-trienoic acid) andB-type, consisting of two epimers having a hydroxyl group at carbon 10(10(S,R)-hydroxy-11(S), 12(S)-epoxy-eicosa-5Z, 8Z,14Z-trienoic acid).

[0170] In the hepoxilin analogs employed in the methods and compositionsof the invention, the epoxide at C11-C12 of the native hepoxilins isreplaced by another group, such as S, —NH or —C_(n)H_(n+2) where n is 1to 4.

[0171] Hepoxilin analogs which may be used in the methods of theinvention comprise compounds of the formula

[0172] wherein X═O, CH₂, S or NH;

[0173] R¹=lower alkyl or alkene;

[0174] lower alcohol (C1 to C22), saturated or unsaturated; or

[0175] —CH₂CH═CH—(CH₂)₃—COR″ wherein R″=OH or O— lower alkyl or alkene;

[0176] R²═OH, NH₂, SH, OPO₃H, lower alkyl or alkene or O— lower alkyl oralkene; and

[0177] R³=lower alkyl or alkene or

[0178] —CH₂—CH═CH—(CH₂)₄—R′″ wherein R′″=CH₃, CH₂OH or CH₂ —O— loweralkyl or alkene or phenyl and substituted phenyl or

[0179] wherein X, R¹, R² and R³ are as in formula I and R⁴ lower alkylor alkene;

[0180] lower alcohol (C1 to C22), saturated or unsaturated; or

—CH═CH—CH₂—CH═CH—(CH₂)₃—COR″

[0181] wherein R″=OH or O— lower alkyl or alkene, and

[0182] derivatives of these compounds, including water solublederivatives of these compounds, such as sugar amides and sugar esters ofthe compounds.

[0183] As used herein, “alkyl” means a branched or unbranched alkylradical. “Lower alkyl or alkene” means C1 to C₂₂ alkyl or alkene.

[0184] Substituted phenyl includes phenyl substituted with —OH, I, Br,Cl or lower alkyl or alkene.

[0185] Preferred hepoxilin analogs are:

[0186] PBT-1 which is8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoic acid methylester, or the corresponding free acid;

[0187] PBT-2 which is8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoic acid methylester, or the corresponding free acid;

[0188] PBT-3 which is10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid methylester, or the corresponding free acid; and

[0189] PBT-4 which is10(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid methylester, or the corresponding free acid. These analogs are described inU.S. Pat. No. 5,616,607, the contents of which are incorporated hereinby reference. Further preferred analogs are the sugar amide and sugarester derivatives of these analogs, including the galactose amides andesters thereof.

[0190] Water-soluble derivatives of the hepoxilins and hepoxilin analogsdescribed herein include sugar amides and sugar esters of the analogs;suitable sugars include monosaccharides such as galactose, glucose andfructose.

[0191] The ability to inhibit thromboxane formation in a mammal usinghepoxilins and hepoxilin analogs described herein enables methods andpharmaceutical compositions useful in the treatment of disordersassociated with an increased or undesirable level of thromboxane or ofthromboxane formation or action, or disorders associated with anundesirable balance between thromboxane and another compound, such as aprostaglandin.

[0192] Such disorders include cardiovascular disease, diabetes mellitus,hypertension, thrombosis and septic shock.

[0193] The specific regulation of arachidonic acid metabolism at varioussteps in its enzymatic metabolism into different products is ofsignificant interest. Total blockade of the whole prostaglandin pathwayis not always desirable. For example, cyclooxygenase inhibitors blockthe whole prostaglandin pathway, eliminating the formation not only ofprostaglandins and thromboxane but also of prostacyclin, a powerfulanti-thrombotic agent. In certain diseases, such as diabetes mellitus,where the ratio of thromboxane to prostacyclin favours thromboxane,diabetic vascular complications can result from such excessivethromboxane formation. What is required in such thromboxane-mediatedconditions is inhibition only of thromboxane formation and not ofprostacyclin or prostaglandin E₂. In fact, enhancing prostacyclinformation could benefit in the control of these vascular complications.

[0194] The present invention provides methods and pharmaceuticalcompositions which inhibit thromboxane formation and therefore serve torestore a more normal thromboxane/prostaglandin balance. This inhibitionis due, at least in part, to inhibition of thromboxane synthase byhepoxilins and hepoxilin analogs as described herein.

[0195] In accordance with a further embodiment, hepoxilins and hepoxilinanalogs inhibit platelet aggregation evoked by thromboxane or by athromboxane analog.

[0196] Collagen activates the release from platelets of arachidonicacid, which is then converted into products via two competing pathways,one through cyclooxygenase to form the prostaglandin endoperoxideintermediate PGH₂, followed in turn by its conversion into thepro-thrombotic unstable compound, thromboxane A₂ (TxA₂ (detected asTxB₂), and the other through 12(S)-lipoxygenase to form the intermediate12(S)-HPETE, which in turn is converted into 12(S)-HETE and the nativehepoxilins.

[0197] It is clear from the data shown herein that the inhibition ofthromboxane-evoked platelet aggregation by hepoxilins and hepoxilinanalogs involves both inhibition of thromboxane formation and antagonismof thromboxane activity mediated through TP receptors, the latter effectbeing seen at lower hepoxilin analog concentrations than the inhibitionof thromboxane formation.

[0198] In a further embodiment, the invention provides methods andcompositions for antagonising thromboxane action, employing thehepoxilins and hepoxilin analogs described herein. The hepoxilin analogPBT-3, for example, shows an affinity for TP receptors similar to thatof the thromboxane agonist U46619, indicating its potential as a potentthromboxane antagonist.

[0199] The hepoxilin analogs described herein have been shown to benon-toxic and well tolerated in in vitro animal studies atconcentrations up to 40 mg/kg (46).

[0200] The invention further enables methods and compositions fortreating eye diseases involving ocular inflammation and/or increasedintra ocular pressure, including glaucoma and diabetic neuropathy. Asdisclosed herein, hepoxilin analogs have been shown to reduce intraocular pressure when administered directly to the eye in the form of eyedrops; the hepoxilin analogs were as effective as the current drug ofchoice for lowering intra ocular pressure, latanoprost. The hepoxilinanalogs also are able to reduce tissue inflammation. Hepoxilins andhepoxilin analogs provide a new family of drugs for the treatment of eyediseases, with the ability both to reduce inflammation and to reduceintra ocular pressure, with the potential to protect against or reverseoptic nerve damage in diseases such as glaucoma and diabetic neuropathy.

[0201] The mechanism of the observed effect of hepoxilins and hepoxilinanalogs on intra ocular eye pressure is presently uncertain but a clearpressure-lowering effect was demonstrated.

[0202] In accordance with the methods and compositions of the presentinvention, one or more hepoxilins or hepoxilin analogs may beadministered to a mammal in a variety of forms depending on the selectedroute of administration, as will be understood by those skilled in theart. The compositions of the invention may be administered orally orparentally, the latter route including intravenous and subcutaneousadministration. Parenteral administration may be by continuous infusionover a selected period of time. Forms for injectable use include sterileaqueous solutions or dispersion and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists.

[0203] In a further embodiment, one or more hepoxilins or hepoxilinanalogs may be administered intra ocularly. Compositions for intraocular use include eye drops comprising the hepoxilin or hepoxilinanalog dissolved in a fluid acceptable for intra ocular administration.A physiological saline may, for example, be used.

[0204] The hepoxilin or hepoxilin analog may be orally administered withan inert diluent or with an assimilable edible carrier, or it may beenclosed in hard or soft shell gelatin capsules, compressed into tabletsor incorporated directly with the food of the diet. For oral therapeuticadministration, a hepoxilin analog may be incorporated with excipientand used in the form in ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions syrups, wafers and the like.

[0205] Compositions containing one or more hepoxilins or hepoxilinanalogs of the present invention can also be administered in a solutionor emulsion contained within phospholipid vesicles called liposomes. Theliposomes may be unilamellar or multilamellar and are formed ofconstituents selected from phosphatidylcholine,dipalmitoylphosphatidylcholine, cholesterol, phosphatidylethanolamtine,phosphatidylserine, dimyristoylphosphatidylcholine and combinationsthereof. The multilamellar liposomes comprise multilamellar vesicles ofsimilar composition to unilamellar vesicles, but are prepared so as toresult in a plurality of compartments in which the analogs containingsolution or emulsion is entrapped. Additionally, other adjuvants andmodifiers may be included in the liposomal formulation such aspolyethyleneglycol, or other materials.

[0206] The liposomes containing the hepoxilin or hepoxilin analogcompositions may also have modifications such as having antibodiesimmobilized on the surface of the liposome in order to target theirdelivery.

[0207] In one embodiment of the present invention is a pharmaceuticalcomposition for administration to subjects in a biologically compatibleform suitable for administration in vivo for treating a disorderassociated with an increased level of thromboxane or a disorder whereinit is desirable to reduce thromboxane activity, including inflammatorydisorders, thrombosis or diabetes and comprising a safe and effectiveamount of a hepoxilin or hepoxilin analog alone, or in combination withother agents and pharmaceutical carriers. The composition may beadministered to any living organism in need of such treatment includinghumans and animals as the composition has efficacy in vivo. By safe andeffective, as used herein, is meant providing sufficient potency inorder to decrease, prevent, ameliorate or treat the disease affectingthe subject while avoiding serious side effects. A safe and effectiveamount will vary depending on the age of the subject, the physicalcondition of the subject being treated, the severity of the disorder,the duration of treatment and the nature of any concurrent therapy, andits determination is within the skill of the ordinary physician.

[0208] A therapeutically active amount of a pharmaceutical compositionof the present invention means an amount effective, at dosages and forperiods of time necessary to achieve the desired result. This may alsovary according to factors such as the disease state, age, sex, andweight of the subject and the ability of the hepoxilin or hepoxilinanalog to elicit a desired response in the subject. Dosage regima may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation.

[0209] By pharmaceutically acceptable carrier as used herein is meantone or more compatible solid or liquid delivery systems. Some examplesinclude but are not limited to starches, sugars, cellulose and itsderivatives, powdered tragacanth, malt, gelatin, collagen, talc, stearicacids, magnesium stearate, calcium sulfate, vegetable oils, polyols,agar, alginic acids, pyrogen free water, isotonic saline, phosphatebuffer, and other suitable non-toxic substances used in pharmaceuticalformulations. Other excipients such as wetting agents and lubricants,tableting agents, stabilizers, anti-oxidants and preservatives are alsocontemplated.

[0210] The compositions described herein can be prepared by knownmethods for the preparation of pharmaceutically acceptable compositionswhich can be administered to subjects, such that an effective quantityof the hepoxilin, hepoxilin analog or analogs is combined in a mixturewith a pharmaceutically acceptable carrier. Suitable carriers aredescribed for example in Remington's Pharmaceutical Sciences (MackPublishing Company, Easton, Pa., USA, 1985). On this basis thecompositions include, albeit not exclusively, solutions of the hepoxilinanalog(s) in association with one or more pharmaceutically acceptablevehicles or diluents, and contained in buffered solutions with asuitable pH and iso-osmotic with the physiological fluids. Hepoxilinanalogs may be prepared as described, for example, in U.S. Pat. No.5,616,607.

EXAMPLES

[0211] The examples are described for the purposes of illustration andare not intended to limit the scope of the invention.

[0212] Methods of chemistry and biochemistry referred td but notexplicitly described in this disclosure and examples are reported in thescientific literature and are well known to those skilled in the art.

[0213] Experimental Procedures

[0214] The following abbreviations are used: TxB₂, thromboxane B₂; HHT,12-hydroxyheptadecatrienoic acid; ADAM, 9-anthryl esters; TLC, thinlayer chromatography; HPLC, high performance liquid chromatography.

[0215] Materials

[0216] The hepoxilin analogs, PBT-1, -2, -3 and -4, were prepared aspreviously described (36); Collagen (Chrono-Par) was purchased fromChrono-log Corp., Havertown, Pa. ADAM reagent (9-anthryldiazomethane)was from Research Organics Inc., Cleveland, Ohio. Tritiated PGH2 andunlabeled PGH2 were from Cayman Chemical, Ann Arbor, Mich. U46619(5-heptenoic acid,7-[6-(3-hydroxy-1-octenyl)-2-oxabicyclo-[2,2,1]-hept-5yl]-[1R-[1α,4α,5β(z),6α(1E,3S*)]-9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F_(2α)),I-BOP (5-heptenoic acid,7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-,[15-[1α, 2α(Z), 3β(1E,3S*), 4α]]) and ¹²⁵I-BOP werefrom Cayman Chemicals, Ann Arbor, Mich., U.S.A.

[0217] Preparation of Human Platelets

[0218] Venous blood was obtained from healthy human subjects who had nottaken NSAID's for at least two weeks. Blood was drawn into plasticsyringes containing ACD. It was immediately centrifuged at 230 at 200×gfor 15 min. The platelet rich plasma was transferred into fresh plastictubes and centrifuged at 400×g for 5 min. The supernatant was discardedand the platelet sediment was resuspended in fresh medium containingNaCl (137 mM), KCl (1 mM), NaH₂PO₄, glucose (5.5 mM), HEPES (20 mM) andCaCl₂ (1 mM) and allowed to stand at room temperature for 30 min. Aplatelet count was made, which determined the volume of plateletsuspension needed to make up 350×10⁶ cells for each measurement. Thealiquot was diluted with medium to make 0.5 ml/assay/cuvette.Appropriate calibration of the aggregometer (PAP4C) for 0% and 100%transmission was carried out through the use of a sample of plateletsuspension and cell-free medium (37).

[0219] Platelet Aggregation and Sample Extraction

[0220] 0.5 ml of platelet suspension was added to siliconized glasstubes (four at a time) and heated with magnetic stirring to 37° for 1min in a platelet aggregometer (PAP-4C). Either vehicle alone (ethanol,1 μl) or hepoxilin analog was then added, followed two min later byagonist (collagen: 2 μg; PGH₂: 100 ng/0.5 m[; U46619: 10 ng/0.5 ml orI-BOP: 2 ng/0.5 ml). The response was recorded for the next 5 min. Inexperiments where analysis of TxB₂ and HHT were to be made by HPLC, theplatelet suspension at the end of the experiment was diluted with ethylacetate, 100 ng of prostaglandin B₁ was added as internal standard, andthe mixture was acidified to pH 3 with 0.1 N HCl. After centrifugation,the organic layer was separated, washed twice with water to neutrality,and evaporated to dryness. Half of the sample was taken and wasresuspended in 1 ml ethyl acetate containing 20 μg ADAM reagent, and wasleft in the dark for 2 h (38). The solvent was then evaporated and theresidue was acetylated with a solution of pyridine/acetic anhydride(3/1, v/v) during 16 h at 23°. The reagents were evaporated to drynessand the residue was resuspended in acetonitrile/water for HPLC analysis.In experiments where the conversion of tritiated PGH₂ was to bemonitored, the procedure described above was repeated with tritiatedPGH₂ instead of unlabeled PGH₂. After extraction of the sample asdescribed above, the radiolabeled compounds were analyzed by TLC. Doseresponse curves for varying amounts of test compounds were generated andthe data expressed as % inhibition of collagen-induced plateletaggregation. Each point was investigated 5 times and statisticalanalysis of the data was carried out using Macintosh Statviewstatistical software.

[0221] In experiments addressing whether levels of endogenously producedthromboxane A2 play a role in the inhibition of aggregation by PBT-3,platelets were treated with aspirin (20 μg/0.5 ml), followed either bycollagen or the thromboxane agonists, I-BOP or U46619; PBT-3 was added,post aspirin but pre-I-BOP or U46619.

[0222] Binding of ¹²⁵I-BOP to Platelets

[0223] Washed platelets were prepared as described above, except thatthe platelet suspension was made up at a concentration of 10×10⁶cells/0.5 ml. The binding assay involved the addition of radioligand(30,000 cpm ¹²⁵I-BOP) to all tubes in triplicate, and either variousdoses of unlabeled I-BOP (10⁻⁹-10⁻⁷ M), PBT-3 (10⁻⁹-10⁻⁷ M) or U46619(10⁻⁹-10⁻⁷ M) in 1 μl ethanol. Additional tubes containing excessunlabeled I-BOP were included to assess the extent of non-specificbinding.

[0224] Measurement of COX-1 and COX-2 Activity

[0225] COX-1 and COX-2 enzyme preparations were purchased from CaymanChemicals. Preliminary studies established that 40 U of COX-1 or COX-2could convert about 70% of ¹⁴C-arachidonic acid (spec. act. 55 mCi/mmol,Ontario Isotopes, Ontario, Canada; 100,000 cpm were diluted with 0.5 μgunlabeled arachidonic acid (Cayman Chemicals)/1 ml assay) into productsin vitro. Different amounts of PBT-3 (10 μg and 20 μg) were added andthe conversion of AA into products was assessed during a 10 min reactionin 1 ml phosphate buffer at 37°. After extraction, products wereassessed by TLC (silica gel G, ethyl acetate/acetic acid, 99/1, v/v).After development, the TLC plates were scanned for radioactive productswith a Berthold TLC radiochromatogram scanner and the radioactivity wasquantified by scraping zones of silica gel, placing in scintillationvials, elution with 1 ml methanol and addition of scintillation medium.Radioactivity was determined by conventional counting in a betascintillation counter.

[0226] Measurements of Platelet Derived Eicosanoids

[0227] Measurement of eicosanoids formed by platelets during treatmentwith collagen or the TP receptor agonists in the presence or absence ofPBT-3 was carried out by HPLC after appropriate derivatization with afluorescent tag (anthracyl diazomethane—ADAM) which forms a fluorescentester (44). The method was adapted to measure the following compounds:TxB2, HHT, 12-HETE and M. The platelet suspension at the end of theexperiment was mixed with ethyl acetate, 100 ng of prostaglandin B₁ wasadded as internal standard and the mixture was acidified to pH 3 with0.1 N HCl. After centrifugation, the organic layer was separated, washedtwice with water to neutrality, and evaporated to dryness. The residuewas resuspended in ethyl acetate and half of the sample was taken. Itwas diluted to 0.2 ml with ethyl acetate containing 20 μg ADAM reagent,and was left in the dark for 2 h. The solvent was then evaporated andthe residue was acetylated with a solution of pyridine/acetic anhydride(3/1, v/v) for 16 h at 23°. The reagents were evaporated to dryness andthe residue was resuspended in acetonitrile/water for HPLC analysis.Dose response curves for varying amounts of test compounds weregenerated and the data were expressed as % inhibition of agonist-inducedplatelet aggregation. Each point was investigated 3 times andstatistical analysis of the data was carried out using MacintoshStatview statistical software.

[0228] Chromatography

[0229] Analysis of the anthryl (ADAM)-acetate derivative of TxB₂, HHTand 12 HETE in the extracted platelet samples was carried out on aHewlett Packard (1100 series) HPLC to which was attached a Shimadzufluorescent detector (RF-10AXL). The detector was operated withexcitation at 364 nm, emission at 411 nm. Chromatographic separation ofthe compound was carried out on a Waters C18 Novapak column (3.9×300 mm)using acetonitrile/water 80/20 at injection with a linear gradient to100% acetonitrile during 20 min. TLC separation of tritiated plateletextracts was carried out on 20 cm silica gel G glass plates (Brinkman),with chloroform/methanol/acetic acid/water (90/9/1/0.65, v/v) asdeveloping solvent in a paper lined glass tank at 230. Afterdevelopment, the radioactive products were detected by scanning the TLCplate on a Berthold TLC radiochromatogram scanner.

[0230] Statistical Analysis

[0231] Values shown are the mean±SD of the number of observations (n)indicated. Analysis of statistical significance was performed usingStudent t-test involving Macintosh Statview software program.

Example 1

[0232] Four hepoxilin analogs (the methyl esters PBT-1, -2, -3 and -4)were tested for inhibition of collagen-evoked aggregation of washedhuman platelet suspensions. FIG. 1 compares the effects of thesecompounds at a single dose (1 μg) added 2 min prior to the addition of apro-aggregatory dose of collagen (2 μg). All four analogs inhibited thecollagen effect, with PBT-3 being the most potent.

[0233]FIG. 2 shows the dose response curves of the four compounds inthis study. The IC50 for the most active compound, PBT-3, was approx.8×10⁻⁷ M. Native hepoxilins were less active than the analogs.

[0234] The galactose amide and galactose ester derivatives correspondingto PBT-1, -2, -3 and 4 were also tested and were found to inhibitcollagen-evoked platelet aggregation (data not shown).

Example 2

[0235] In order to determine whether the hepoxilin analogs affect TxA₂formation (measured as the stable degradation product TxB2), experimentswere set up in which the contents of the cuvettes from aggregationexperiments as described above were extracted for TxB₂ and analyzed byHPLC after appropriate derivatization of the samples for fluorescencedetection. The results are shown in FIG. 3. TxB₂ formation was inhibitedin a dose-dependent manner by the four analogs, again to a greaterextent by PBT-3. The IC₅₀ for inhibition of TxB2 formation was in thesame range as that for inhibition of platelet aggregation.

Example 3

[0236] Additional experiments were set up to directly monitor theinhibitory effects of PBT-3 on thromboxane formation. PGH₂ is convertedinto TxB₂ by platelets. As shown in FIG. 4, PGH₂ at 100 ng causedplatelets to aggregate. Addition of PBT-3 at various doses beforeaddition of PGH₂ inhibited the second wave of aggregation which is knownto be evoked by formation of TxA₂, a potent pro-aggregating substance.This is additional confirmation that PBT-3 inhibited TxA₂ formation.

Example 4

[0237] Tritiated PGH₂ was employed to confirm its conversion into TxB₂by platelet suspensions and the inhibition of this formation of TxA₂ byPBT-3. FIG. 5 shows a TLC profile demonstrating the inhibitory action oftwo doses of PBT-3 on TxB2 formation (as a measure of the bioactiveproduct TxA₂).

Example 5

[0238] Additional experiments were set up to test the actions of thehepoxilin analogs on platelet aggregation evoked by the TxA₂ mimic,U46619, working through the TP receptor. The methods were as in Example3, but instead of collagen, U46619 was added to evoke aggregation. PBT-3at a dose of 1 μg inhibited the aggregation by U46619 (FIG. 6).

Example 6

[0239] Dose related aggregation curves for I-BOP, a potent thromboxanereceptor agonist, indicated that at a concentration of 2 ng/0.5 ml, itcaused 70% aggregation (data not shown). This dose was therefore chosenas it represented a point at which inhibition curves for the testcompounds could be most sensitive (FIG. 7A). Addition of PBT-3 2 minutesprior to I-BOP caused a pronounced inhibition of I-BOP-inducedaggregation in a dose related fashion. FIG. 7A shows aggregationresponses in human washed platelet suspensions evoked by I-BOP and theinhibition of this response by different doses of PBT-3 added 2 minprior to the addition of I-BOP. FIG. 7B shows aggregation responses ofhuman washed platelets challenged with U46619 and their inhibition bydifferent amounts of PBT-3. FIG. 8 provides quantitative data for theseexperiments demonstrating an IC₅₀ for inhibition of aggregation evokedby I-BOP at 0.6×10⁻⁷ M of PBT-3. PBT-3 also inhibited the aggregationevoked by the agonist, U46619 with an IC₅₀ of 7×10⁻⁷ M demonstrating thegreater selectivity of PBT-3 for I-BOP versus U46619 (FIG. 8). Collagenevokes the aggregation of human platelets through the formation ofthromboxane A₂. PBT-3 dose dependently prevented collagen-evokedaggregation of platelets with an IC₅₀ of 8×10⁻⁷ M. Analysis of thethromboxane formed in these experiments indicated that collagen-evokedformation of thromboxane was blocked by PBT-3 with an IC₅₀ of 8×10⁻⁷ M.In separate studies (data not shown) it was demonstrated thatI-BOP-evoked or U46619-evoked aggregation was not accompanied bythromboxane formation; hence PBT-3 inhibition of the action of these twothromboxane mimetics is due to direct inhibition at the TP receptorlevel and was not dependent on endogenous formation (or blockade) ofthromboxane A₂.

Example 7

[0240] The binding of ¹²⁵I-BOP to platelets was examined, as Well as thecompetition of I-BOP, PBT-3 and U46619 with this binding. FIG. 9 showsthat PBT-3 competed with the binding of ¹²⁵I-BOP to platelets in a dosedependent way. Competition curves are shown for I-BOP (IC₅₀ 0.5×10⁻⁹ M),PBT-3 (IC₅₀=8.1×10⁹ M) and U46619 (IC₅₀=4.1×10⁻⁹ M). PBT-3 was about 16fold less active than I-BOP in competing with ¹²⁵I-BOP binding to theplatelet TP receptor, but was of similar activity to U46619:

Example 8

[0241] To establish whether inhibition of endogenous formation ofthromboxane plays a role in the inhibitory effect of PBT-3 on theplatelet aggregation process, rather than antagonism of thromboxaneaction at the TP receptor, experiments were carried out in whichplatelets were treated with aspirin (to block endogenous thromboxaneformation) followed by the agonist, U46619 (which causes aggregationeven after aspirin treatment). FIG. 10 shows an aggregation profile ofsuch an experiment. Both collagen and U46619 caused platelets toaggregate—see FIG. 10, lines 1 and 2 respectively. Aspirin greatlyreduced collagen-evoked aggregation (FIG. 10—early part of line 3). Adose of 20 μg aspirin was employed for this study, as this dose blockedcollagen effects almost completely (data not shown), demonstrating thatcollagen-induced platelet aggregation is mediated through the formationof endogenous thromboxane. In contrast, aspirin at this dose did notblock U46619-evoked aggregation (FIG. 10—later part of line 3)demonstrating that aspirin did not interfere with the TP receptor or thecascade of events initiated by U46619. Addition of PBT-3 toaspirin-treated platelets before the addition of U46619 resulted in ablockade of the aggregation induced by U46619 (FIG. 10—line 4, comparewith line 3). This confirmed that PBT-3 caused inhibition of the actionof U46619 at the TP receptor level, independently of its effects on theformation of endogenous thromboxane.

Example 9

[0242] Several experiments were carried out to investigate whether PBT-3affected several enzymes involved in the generation of variouseicosanoids, or whether it acted to block selectively thromboxaneformation subsequent to its actions at the TP receptor. The data aresummarized in Table 1. Neither COX-1, COX-2,12-LOX nor Plase A2 wasinhibited by relatively large amounts of PBT-3, i.e. about 3-4 log dosesgreater than that required to inhibit the TP receptor. In contrast,PBT-3 significantly inhibited TxB2 formation in platelets (Table 1, lastcolumn).

Example 10

[0243] Brown Norway rats (300 g) were purchased from Charles River (St.Constant, Que), and housed (two/cage) in an animal facility with freeaccess to food and water. A light cycle of 12 h on and 12 h off wasmaintained. Animals were allowed to adjust for one week prior totreatment during which period they were handled daily to get themfamiliarized with the procedures.

[0244] Prior to treatment with test drugs, the rats' intra ocularpressures (IOP) were measured to establish baseline readings. One dropof Didocaine (0.5%, Dioptic Labs, Markham, Ont) was administered to eacheye, and the pressure in each eye was measured with a TonoPen (MentorXL, Innova Medical Ophthalmics, Toronto). The treatment protocolconsisted of daily administration of one drop of one of the followingsubstances to the left eye, the right eye being left untreated to act asa control. Each animal received one of the following treatments dailyfor three weeks: Ophtho-bunolol (0.5%)+latanoprost (Xalatan) (0.005%),latanoprost (0.005%), normal saline, PBT-3 in saline (0.005%), PBT-30(10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z, 8Z, 14Z-trienoic acidgalactosamide) in saline (0.005%), or Ophtho-bunolol (0.5%). IOP wasmeasured weekly. Following this, the animals were left without treatmentfor 2 weeks (wash-out), and then treatment was started again, the sameanimals receiving the same drugs. Results are calculated from at least30 readings/test animal and presented as the mean±SEM (left eye vs righteye), with the exclusion of readings >50 and <10 which were consideredspurious and artifactual readings.

[0245]FIG. 11 is representative of one measurement point after 10 days'treatment. While latanoprost gave a significant reduction in IOP,O-bunolol gave a non-significant rise, which is also reflected in areduced effect of the combination of latanoprost and O-bunolol. PBT-3gave a significant reduction in IOP, comparable to that produced bylatanoprost. PBT-30 also tended to reduce IOP but the effect did notreach statistical significance at the dose tested.

[0246] Saline, the vehicle for the PBT compounds, caused an increase inIOP relative to the untreated eye (data not shown).

Example 11

[0247] Galactose amides of hepoxilin analogs were prepared as follows.

[0248] All solvents were glass distilled and used as obtained (Caledon,Georgetown, Ontario). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide(DMAPEC), N-hydroxysuccinimide and D-galactosamine.HCl were obtainedfrom Aldrich Chemicals (USA).

[0249] 8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z, 9E, 14Z-trienoic methylester (compound (1) of FIG. 12) was prepared as described previously inU.S. Pat. No. 5,616,607. The corresponding free acid was prepared byconventional hydrolysis of the methyl ester, in NaOH/ethanol for 30minutes at 23° and extraction with ethyl acetate after acidification.The resulting free acid was purified by HPLC by conventional techniquesand subjected to activation as described below.

[0250] 30 μmol of the free acid was coupled, through the free carboxylgroup, with N-hydroxysuccinimide (220 μmol) in methylene chloride.Finely ground activated molecular sieves (3A) were added, followed bythe addition of 220 μmol of DMAPEC. The reaction mixture was stirred for3 h at 20°, the molecular sieves were filtered off, and the solution waswashed with water (3×2 ml) and taken to dryness. The residue waspurified by preparative RP-HPLC (Nova-Pak C18 column (Waters), 3.9×300mm, eluent acetonitrile-water, 70:30, flow rate 1.5 ml/min, UV detectorat 205 nm) and the fraction with retention time 5.0-5.2 minutes wascollected. After evaporating the solvents, a colourless oil was obtainedwith a yield of approximately 89-93%. Electrospray mass spectrometry(API III Plus triple quadrupole with direct injection) was done toconfirm the structure of the N-hydroxy succinimide ester (compound (2)of FIG. 12) with [M+NH₄]⁺ at 449 (100% intensity).

[0251] The N-hydroxysuccinimide ester (28 μmol) was then dissolved in1,4-dioxane. A solution of 140 μmol of D-galactosamine.HCl and 180 μmolof NaHCO₃ in water was added, and the mixture was allowed to stand for 4h at 200. The solvents were taken to dryness and the residue waspurified by preparative RP-HPLC (acetonitrile-water, 50:50; flow rate1.0 ml/min). The fraction between 7.0-7.5 minutes was collected. Afterevaporating the solvents, white crystals were obtained with a yield ofapproximately 79-83%. The structure was confirmed by NMR and massspectrometry (m/z 513 [M+NH₄]⁺; 100% intensity).

Example 12

[0252] Synthesis of 1,2,3,4-tetra(2,6-di-O-tert-butyldimethylsilyl)Protected Galactose Intermediate (1)

[0253] Galactose esters of hepoxilins and hepoxilin analogs weresynthesised as described in Demin et al. (45), and as described brieflybelow. The general synthetic scheme is shown in FIGS. 13 and 14.

[0254] To obtain the 1,2,3,4-tetraBDMS(1,2,3,4-tetra(2,6-di-O-tert-butyldimethylsilyl)) protected galactose 1(FIG. 13), diisopropylidene protected galactose 2 was acetylated toyield compound 3, followed by acetonide deprotection of compound 3 with90% TFA in water to yield 6-acetyl galactose 4. 6-acetyl galactose 4 wassilylated using BDMSCI-imidazole-DMF. Basic hydrolysis of the obtained6-acetate 5 led to the target crystalline material,1,2,3,4-tetra-O-(tert-butyldimethylsilyl)-D-galactopyranose 1, obtainedas a mixture of α- and β-anomers (FIG. 13).

[0255] 1,2:3,4-Di-O-isopropylidene-6-acetyl-α-D-galactopyranose (3)

[0256] To 520 mg (2.0 mmol) of1,2:3,4-Di-O-isopropylidene-D-galactopyranose 2 (R_(f) 0.06,EtOAc-hexane, 1:2) 2.0 mL pyridine and 1.5 mL. acetic anhydride wereadded. The reaction mixture was kept at 60° C. for 3 h and the reagentswere removed with a stream of nitrogen. The yield of 6-acetate 3 was 580mg (96%). R_(f) 0.22, EtOAc-hexane, 1:2. The compound was used withoutfurther purification. Distillation on a kugelrohr apparatus at an oventemperature of 120° C. and under a 0.1 mm Hg vacuum gave an analyticalsample as a viscous oil.

[0257] [α]_(D) ²⁵-47° (c 1.0, CHCl₃). ¹H-NMR (CDCl₃, δ, ppm): 1.34,1.45, 1.52, 1.59 (4×s, 12H, isopropylidene), 2.09 (s, 3H, OAc), 4.03 (m,1H, H⁵), 4.19 (dd, 1H, J 7.5 and 11.5 Hz, H⁶), 4.24 (br. d, 1H, J 7.9Hz, H⁴), 4.29 (dd, 1H, J 4.9 and 11.5 Hz, H⁶′), 4.33 (dd, 1H, J 2.3 and4.9 Hz, H²), 4.62 (dd, 1H, J 2.3 and 7.9 Hz, H³), 5.54 (d, 1H, J 4.9 Hz,H¹). MS (m/e, relative intensity, %): 303 ([M+H]⁺, 100), 320 (M+NH₄]⁺,97).

[0258] 6-Acetyl-α,β-D-galactopyranose (4)

[0259] To 580 mg (1.92 mmol) of1,2:3,4-Di-O-isopropylidene-6-acetyl-D-galactopyranose 3 2.0 mL of 90%trifluoroacetic acid was added. The reaction mixture was kept at 20° C.for 15 min and the reagents were removed with a stream of N₂ followed byazeotropic evaporation of traces of water with acetonitrile (3×20 mL).The yield of 6-acetyl-D-galactopyranose 4 was 392 mg (92%). The compoundwas crystallized from 5% MeOH in MeCN, m.p. 139-141° C. (lit^(l), m.p.130-132° C.).

[0260] [α]_(D) ²⁵+91° (c 1.0, H₂O) (lit^(l), [α]_(D) ²⁵+95.6° (c 1.0,MeOH). MS (m/e, relative intensity, %): 204 ([M−H₂O]⁺, 60), 223 ([M+H]⁺,33), 240 ([M+NH₄]⁺, 100).

[0261]1,2,3,4-Tetra-O-(t-butyldimethylsilyl)-6-acetyl-α,β-D-galactopyranose(5)

[0262] 6-Acetyl-D-galactopyranose 4 (392 mg, 1.76 mmol) was dissolved in30 mL DMF. t-BDMSCI (1590 mg, 10.56 mmol) and imidazole (1440 mg, 21.12mmol) were added. The reaction mixture was stirred at 50° C. for 12 h,poured into 200 mL of water and extracted with hexane (3×50 mL). Thehexane fractions were dried with Na₂SO₄ and evaporated. The residue wasdistilled on a Kugelrohr apparatus at 0.01 mm Hg, collecting a fractionat an oven temperature of 170-180° C. The obtained colorless oil waspurified by column chromatography on silica gel, eluent 2-5% EtOAc inhexane, and the target fraction was collected (R_(f) 0.63, EtOAc-hexane,1:2). After evaporation a viscous oil 5 was obtained (780 mg, 65%).

[0263] [α]_(D) ²⁵−40° (c 0.5, CHCl₃). ¹H-NMR (CDCl₃, 6, ppm): 0.09, 0.90(2×m, 60H, Si^(t)Bu and SiMe₂), 2.05 (s, 3H, OAc), 3.90-4.03 (m, 5H,H^(2+4+5+6+6′)), 4.17 (dd, 1H, J 3.9 and 11.4 Hz, H³), 5.14 (br. s., 1H,H¹). MS (m/e, relative intensity, %): 547 ([M+H−BDMSOH]⁺, 100), 679([M+H]⁺, 51), 696 ([M+NH₄]⁺, 82).

[0264] 1,2,3,4-Tetra-O-(tert-butyldimethylsilyl)-α,β-D-galactopyranose(1)

[0265]1,2,3,4-Tetra-O-(tert-butyldimethylsilyl)-6-acetyl-D-galactopyranose 5(460 mg, 0.68 mmol) was dissolved in 150 mL THF-MeOH, 1:1. 20 mL 1 NNaOH was added and the reaction mixture was stirred for 8 h at 20° C.The organic solvents were evaporated, 200 mL of saturated NaCl solutionwas added and the mixture was extracted with benzene (4×100 mL). Theorganic layers were combined and dried with Na₂SO₄, taken to dryness andpurified by column chromatography on silica gel using 2-20% EtOAc inhexane as the eluent. The fraction with R_(f) 0.47 (EtOAc-hexane, 1:2)was collected. After evaporation the target compound 1 (crystals, 337mg, 78%) was obtained. The analytical sample was crystallized fromhexane at −70° C. giving white needle-shape crystals, m.p. 130-132° C.

[0266] [α]_(D) ²⁵−57° (c 1.0, CHCl₃). ¹H-NMR (CDCl₃, δ, ppm): 0.11, 0.90(2×m, 60H, Si^(t)Bu and SiMe₂), 3.64 (dd, 1H, J 5.1 and 9.8 Hz, H⁴),3.70 (dd, 1H, J 1.7 and 9.8 Hz, H³), 3.78 (m, 1H, H⁵), 3.85 (br.s, 1H,H²), 4.06, 4.13 (2×m, 2H, H^(6+6′)), 5.28 (br.s, 1H, H¹). MS (m/e,relative intensity, %): 523 ([M+H−BDMSOH+NH₄]⁺, 55), 540([M−BDMSOH+NH₄+NH₄]⁺, 100), 637 ([M+H]⁺, 3.7), 654 ([M+NH₄]⁺, 4.5).

[0267] Synthesis of Galactose Esters of Hepoxilins

[0268] As shown in FIG. 14,1,2,3,4-Tetra-O-(tert-butyldimethylsilyl)-α,β-D-galactopyranose 1 isreacted with 8 or 10-BDMS protected hepoxilins 7a,b in the presence ofEDAC/DMAP (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(EDAC)/4-dimethylaminopyridine (DMAP)) to give the hepoxilinyl esters oftetraBDMS galactose 8a,b. The anomers of tetraBDMS galactose fattyesters 8a,b were separable both by TLC and HPLC. The treatment of thecompounds 8a,b with equimolar amounts of n-Bu₄NF, Py.HCl, THF gave thetarget compounds 9a,b. Both α- and β-anomers of the hepoxilin esters8a,b led to the same products 9a,b after deprotection. RP-HPLC analysisof the products 9a,b showed a poorly separated doublet, corresponding tothe mutarotating α- and β-anomers (FIG. 13).

[0269] 8 or 10-tert-Butyldimethylsilyl-8 or 10S/R-delta-HxA₃ anddelta-HxB₃ (7a_(s,r), 7b_(s,r))

[0270] To 4 mg (11.5 μmol) of the corresponding delta-Hx methyl ester, asolution of 15 mg (100 μmol) of t-butyldimethylchlorosilane and 13.6 mg(200 μmol) of imidazole in 100 μl DMF was added. The mixture was kept at50° C. for 0.5 h, 5 mL of water was added and the mixture was extractedwith hexane (3×2 mL). After removing the hexane from the organic layer,the residue was purified by RP-HPLC (column 3.9×300 mm, 100% MeCN, flowrate 2.0 mL/min). The fraction having a retention time of 6.5 min wascollected. A colorless oil was obtained after evaporation of thesolvent. The yield of the 8 or 10-tert-butyldimethylsilyl derivatives ofdelta-Hx methyl esters was 5.3 mg (100%). The methyl esters werehydrolyzed to 8/10-tert-butyldimethylsilyl-(8/10R/S)-delta-HxA₃/B₃(MeOH-1N NaOH, 2:1, 4 h, RT). After acidification to pH 4, the mixturewas extracted with EtOAc and passed through silica gel using EtOAc asthe eluent. After evaporation the yield of BDMS-derivatives of delta-Hx7a,b was 5.15 mg (100% after two steps).

[0271] 1,2,3,4-Tetra-O-(t-butyldimethylsilyl)-6-[8 or10-t-butyidimethylsilyl]delta-[8 or 10S/R]-hepoxilinA₃(B₃)-yl-α,β-D-galactopyrahoses (8a_(s,r), 8b_(b,r)) 5.15 mg (11.5μmol) of 10-BDMS derivatives of delta-Hx 7a,b in 3 mL CH₂Cl₂ weretreated with 10.9 mg (17.3 μmol) oftetra-O-(t-butyldimethylsilyl)-D-galactopyranose 1 6.6 mg (34.5 μmol) ofEDAC and 0.1 mg of DMAP for 24 h at 20° C. HPLC analysis (column 3.9×75mm, 100% MeCN, flow rate 5.0 mL/min) showed two peaks in a 1:1 ratio,and retention times 11.6 and 14.6 min corresponding to the α- andβ-anomers. The mixture was washed with water (2×2 mL), taken to drynessand purified by RP-HPLC in the same system, collecting both α- andβ-anomers as a single fraction. Total yield 9.9-10.1 mg (80-82%).

[0272]¹H-NMR (CDCl₃, δ, ppm), 8a_(s), 8a_(r) (identical to each other,see also ref.⁵): 0.53 (m, 2H, cyclopropyl), 0.76, 1.16 (2×m, 2×1H, H¹¹and H¹² in the fatty chain), 4.02 (dt, 1H, J 6.1 and 6.4 Hz, H⁸ in thefatty chain), 5.23, 5.25 (2×br. s, 2×0.5H, H^(1α) and H^(1β)); 0.10,0.90 (BDMS); 0.90, 1,27-1.38, 1.70, 2.01, 2.06, 2.20, 2.33, 5.37-5.45(fatty chain moiety); 3.61, 3.67-3.76, 3.80, 3.86, 3.96, 4.14, 4.23,4.84, 5.09, 5.14 (α- and β-galactose moiety). 8b_(s): identical to8a_(s), 8a_(r) except the following: 0.23, 0.41 (2×m, 2×1H,cyclopropyl), 0.71-0.80 (m, 2H, H¹¹⁺¹² in the fatty chain), 3.97 (m, 1H,H¹⁰). 8b_(r): identical to 8b_(s) except the following: 3.86 (m, 1H,H¹⁰). MS (either regio/stereoisomer, m/e, relative intensity, %): 970([M−BDMSOH+NH₄+NH₄]⁺, 100)

[0273] 6-(8 or 10S/R)-Hepoxilin A₃(B₃)-yl-α,β-D-galactopyranoses(9a_(s,r), 9b_(s,r)) to 3 mg (2.81 μmol) of the compounds 8a,b in 300 μlof freshly distilled THF, 40 μl of 0.5M Py.HCl in THF-5% H₂O was added,followed by 20 μl of 1 M n-Bu₄NF in THF. The mixture was allowed tostand for 0.5 h at 20° C., the THF was removed, and the residue waspurified by RP-HPLC (column 3.9×300 mm, MeCN—H₂O, 60:40, flow rate 1.0mL/min). The doublet of peaks at retention times 6.0-7.0 min wascollected. The yield was 1.10 mg (79%). MS (either regio/stereoisomer,m/e, relative intensity, %): 461 ([M+H—H₂O—H₂O]+), 479 (M+H—H₂O]⁺), 514([M+NH₄]⁺, 100). TABLE 1 Effect of PBT-3 on various enzyme systemsrelated to arachidonic acid metabolism Enzyme activity (n = 3)Concentration (% of control values, mean ± SD) of PBT-3 (μM) COX-1*COX-2* Plase A₂ ⁺ 12-LOX⁺ TX 30.0 116.8 ± 11^(NS) 107.1 ± 14^(NS) ND NDND 3.00 119.6 ± 14^(NS) 109.2 ± 8.0^(NS) 120.8 ± 18.3^(NS) 102.5 ±5.7^(NS) 29.9 ± 6.7** 0.60 ND ND  89.5 ± 15.6^(NS)  73.0 ± 4.9** 41.0 ±4.7** 0.30 ND ND  91.9 ± 5.6^(NS)  85.2 ± 7.4^(NS) 65.7 ± 6.0* 0.05 NDND 100.0 ± 6.9^(NS)  79.4 ± 7.6* 94.2 ± 15.2^(NS) # products includingHETEs and prostaglandins.

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We claim:
 1. A method for inhibiting thromboxane formation in a mammalcomprising administering to the mammal an effective amount of ahepoxilin or of a hepoxilin analog of the formula:

wherein X is O, CH₂, S or NH; R¹ is lower alkyl or alkene; lower alcohol(C1 to C22), saturated or unsaturated; or —CH₂CH═CH—(CH₂)₃—COR″ whereinR″ is OH, O— lower alkyl or alkene; R² is OH, NH₂, SH, OPO₃H, loweralkyl or alkene or O— lower alkyl or alkene; and R³ is lower alkyl oralkene or —CH₂—CH═CH—(CH₂)₄—R′″ wherein R′″ is CH₃, CH₂OH, CH₂ —O— loweralkyl or alkene, phenyl or substituted phenyl or

wherein X, R¹, R² and R³ are as defined for formula I and R⁴ is loweralkyl or alkene; lower alcohol (C1 to C22), saturated or unsaturated; or—CH═CH—CH₂—CH═CH—(CH₂)₃—COR″ wherein R″=OH or O— lower alkyl or alkene,or of a derivative thereof.
 2. The method of claim 1 wherein substitutedphenyl is phenyl substituted with OH, I, Br, Cl or lower alkyl oralkene.
 3. A method for inhibiting thromboxane formation in a mammalcomprising administering to the mammal an effective amount of a compoundselected from the group consisting of: (a)8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoic acid or aderivative thereof; (b)8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoic acid or aderivative thereof; (c)10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof; and (d)10(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof.
 4. The method of any one of claims 1 to 3 whereinthe compound is administered in the form of a derivative of the trienoicacid selected from the group consisting of a methyl ester, a sugar amideand a sugar ester.
 5. The method of any one of claims 1 to 3 wherein thecompound is administered in the form of a galactose ester or a galactoseamide.
 6. The method of any one of claims 1 to 5 wherein the mammalsuffers from a disorder selected from the group consisting ofcardiovascular disease, thrombosis, diabetes mellitus and septic shock.7. A method for antagonising thromboxane activity in a mammal comprisingadministering to the mammal an effective amount of a hepoxilin or of ahepoxilin analog of the formula:

wherein X is O, CH₂, S or NH; R¹ is lower alkyl or alkene; lower alcohol(C1 to C22), saturated or unsaturated; or —CH₂CH═CH—(CH₂)₃—COR″ whereinR″ is OH, O— lower alkyl or alkene; R² is OH, NH₂, SH, OPO₃H, loweralkyl or alkene or O— lower alkyl or alkene; and R³ is lower alkyl oralkene or —CH₂—CH═CH—(CH₂)₄—R″ wherein R′″ is CH₃, CH₂OH, CH₂—O— loweralkyl or alkene, phenyl or substituted phenyl or

wherein X, R¹, R² and R³ are as defined for formula I and R⁴ is loweralkyl or alkene; lower alcohol (C1 to C22), saturated or unsaturated; or—CH═CH—CH₂—CH═CH—(CH₂)₃—COR″ wherein R″=OH or O— lower alkyl or alkene,or of a derivative thereof.
 8. The method of claim 7 wherein substitutedphenyl is phenyl substituted with OH, I, Br, Cl or lower alkyl oralkene.
 9. A method for antagonising thromboxane activity in a mammalcomprising administering to the mammal an effective amount of a compoundselected from the group consisting of: (a)8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoic acid or aderivative thereof; (b)8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoic acid or aderivative thereof; (c)10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof; and (d)10(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof.
 10. The method of any one of claims 7 to 9 whereinthe compound is administered in the form of a derivative of the trienoicacid selected from the group consisting of a methyl ester, a sugar amideand a sugar ester.
 11. The method of any one of claims 7 to 9 whereinthe compound is administered in the form of a galactose ester or agalactose amide.
 12. The method of any one of claims 7 to 11 wherein themammal suffers from a disorder selected from the group consisting ofcardiovascular disease, thrombosis, diabetes mellitus and septic shock.13. A method of preventing or reducing thromboxane-mediated plateletaggregation in a mammal comprising administering to the mammal aneffective amount of a hepoxilin or of a hepoxilin analog of the formula:

wherein X is O, CH₂, S or NH; R¹ is lower alkyl or alkene; lower alcohol(C1 to C22), saturated or unsaturated; or —CH₂CH═CH—(CH₂)₃—COR″ whereinR″ is OH, O— lower alkyl or alkene; R² is OH, NH₂, SH, OPO₃H, loweralkyl or alkene or O— lower alkyl or alkene; and R³ is lower alkyl oralkene or —CH₂—CH═CH—(CH₂)₄—R′″ wherein R′″ is CH₃, CH₂OH, CH₂ —O— loweralkyl or alkene, phenyl or substituted phenyl or

wherein X, R¹, R² and R³ are as defined for formula I and R⁴ is loweralkyl or alkene; lower alcohol (C1 to C22), saturated or unsaturated; or—CH═CH—CH₂—CH═CH—(CH₂)₃—COR″ wherein R″=OH or O— lower alkyl or alkeneor a derivative thereof.
 14. A method of preventing or reducingthromboxane-mediated platelet aggregation in a mammal comprisingadministering an effective amount of a compound selected from the groupconsisting of: (a)8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoic acid or aderivative thereof; (b) 8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z, 10E,14Z-trienoic acid or a derivative thereof; (c)10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof; and (d)10(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof.
 15. The method of claim 13 or 14 wherein thecompound is administered in the form of a derivative of the trienoicacid selected from the group consisting of a methyl ester, a sugar amideand a sugar ester.
 16. A method of treating a thromboxane-mediateddisease in a mammal comprising administering to the mammal an effectiveamount of a hepoxilin or of a hepoxilin analog of the formula:

wherein X is O, CH₂, S or NH; R¹ is lower alkyl or alkene; lower alcohol(C1 to C22), saturated or unsaturated; or —CH₂CH═CH—(CH₂)₃—COR″ whereinR″ is OH, O— lower alkyl or alkene; R² is OH, NH₂, SH, OPO₃H, loweralkyl or alkene or O— lower alkyl or alkene; and R³ is lower alkyl oralkene or —CH₂—CH═CH—(CH₂)₄—R′″ wherein R′″ is CH₃, CH₂OH, CH₂ —O— loweralkyl or alkene, phenyl or substituted phenyl or

wherein X, R¹, R² and R³ are as defined for formula I and R⁴ is loweralkyl or alkene; lower alcohol (C1 to C22), saturated or unsaturated; or—CH═CH—CH₂—CH═CH—(CH₂)₃—COR″ wherein R″=OH or O— lower alkyl or alkeneor a derivative thereof.
 17. A method of treating a thromboxane-mediateddisease in a mammal comprising administering to the mammal an effectiveamount of a compound selected from the group consisting of: (a)8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E, 14Z-trienoic acid or aderivative thereof; (b) 8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z, 10E,14Z-trienoic acid or a derivative thereof; (c)10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof; and (d)10(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof.
 18. The method of claim 16 or 17 wherein the diseaseis a vascular disease.
 19. A method for treating or preventing an eyedisease associated with 5 ocular inflammation and/or increasedintraocular pressure comprising administering to the mammal an effectiveamount of a hepoxilin or of a hepoxilin analog of the formula:

wherein X is O, CH₂, S or NH; R¹ is lower alkyl or alkene; lower alcohol(C1 to C22), saturated or unsaturated; or —CH₂CH═CH—(CH₂)₃—COR″ whereinR″ is OH, O— lower alkyl or alkene; R² is OH, NH₂, SH, OPO₃H, loweralkyl or alkene or O— lower alkyl or 20 alkene; and R³ is lower alkyl oralkene or —CH₂—CH═CH—(CH₂)₄—R′″ wherein R′″ is CH₃, CH₂OH, CH₂ —O— loweralkyl or alkene, phenyl or substituted phenyl or

wherein X, R¹, R² and R³ are as defined for formula I and R⁴ is loweralkyl or alkene; lower alcohol (C1 to C22), saturated or unsaturated; or—CH═CH—CH₂—CH═CH—(CH₂)₃—COR″ wherein R″=OH or O— lower alkyl or alkeneor a derivative thereof.
 20. A method for treating or preventing an eyedisease associated with ocular inflammation and/or increased intraocularpressure comprising administering to the mammal an effective amount of acompound slected from the group consisting of: (a)8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z, 10E, 14Z-trienoic acid or aderivative thereof: (b) 8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoic acid or a derivative thereof; (c)10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof; and (d)10(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof.
 21. The method of claim 19 or 20 wherein the eyedisease is glaucoma.
 22. The method of claim 19 or 20 wherein the eyedisease is diabetic neuropathy.
 23. A method for reducing intra-ocularpressure in a mammal comprising administering intra-ocularly to themammal an effective amount of at least one of a hepoxilin or of ahepoxilin analog of the formula:

wherein X is O, CH₂, S or NH; R¹ is lower alkyl or alkene; lower alcohol(C1 to C22), saturated or unsaturated; or —CH₂CH═CH—(CH₂)₃—COR″ whereinR″ is OH, O— lower alkyl or alkene; R² is OH, NH₂, SH, OPO₃H, loweralkyl or alkene or O— lower alkyl or alkene; and R³ is lower alkyl oralkene or —CH₂—CH═CH—(CH₂)₄—R′″ wherein R′″ is CH₃, CH₂OH, CH₂ —O— loweralkyl or alkene, phenyl or substituted phenyl or

wherein X, R¹, R² and R³ are as defined for formula I and R⁴ is loweralkyl or alkene; lower alcohol (C1 to C22), saturated or unsaturated; or—CH═CH—CH₂—CH═CH—(CH₂)₃—COR″ wherein R″=OH or O— lower alkyl or alkeneor a derivative thereof.
 24. A method for reducing intra-ocular pressurein a mammal comprising administering intra-ocularly to the mammal aneffective amount of a compound selected from the group consisting of:(a) 8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoic acid or aderivative thereof; (b) 8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z, 10E,14Z-trienoic acid or a derivative thereof; (c)10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof; and (d)10(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof.
 25. The method of any one of claims 19 to 24 whereinthe compound is administered in the form of a derivative of the trienoicacid selected from the group consisting of a methyl ester, a sugar amideand a sugar ester.
 26. The method of any one of claims 19 to 24 whereinthe compound is administered in the form of a galactose ester.
 27. Useof a compound of the formula:

wherein X is O, CH₂, S or NH; R¹ is lower alkyl or alkene; lower alcohol(C1 to C22), saturated or unsaturated; or —CH₂CH═CH—(CH₂)₃—COR″ whereinR″ is OH, O— lower alkyl or alkene; R² is OH, NH₂, SH, OPO₃H, loweralkyl or alkene or O— lower alkyl or alkene; and R³ is lower alkyl oralkene or —CH₂—CH═CH—(CH₂)₄—R′″ wherein R′″ is CH₃, CH₂OH, CH₂ —O— loweralkyl or alkene, phenyl or substituted phenyl or

wherein X, R¹, R² and R³ are as defined for formula I and R⁴ is loweralkyl or alkene; lower alcohol (C1 to C22), saturated or unsaturated; or—CH═CH—CH₂—CH═CH—(CH₂)₃—COR″ wherein R″=OH or O— lower alkyl or alkene,or a derivative thereof, for the preparation of a medicament for atreatment selected from the group consisting of: (a) for inhibitingthromboxane formation in a mammal; (b) for treating or preventing eyedisease associated with ocular inflammation and/or increased intraocularpressure in a mammal; (c) for inhibiting thromboxane activity in amammal; (d) for preventing or reducing thromboxane-mediated plateletaggregation in a mammal; (e) for treating a thromboxane-mediated diseasein a mammal; and (f) for reducing intra-ocular pressure in a mammal. 28.Use of a compound selected from the group consisting of: (a)8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoic acid or aderivative thereof; (b)8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoic acid or aderivative thereof; (c)10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof; and (d)10(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof, for a treatment selected from the group consistingof: (a) for inhibiting thromboxane formation in a mammal; (b) fortreating or preventing eye disease associated with ocular inflammationand/or increased intraocular pressure in a mammal; (c) for inhibitingthromboxane activity in a mammal; (d) for preventing or reducingthromboxane-mediated platelet aggregation in a mammal; (e) for treatinga thromboxane-mediated disease in a mammal; and (f) for reducingintra-ocular pressure in a mammal.
 29. A composition for application tothe eye comprising as active ingredient a compound of the formula:

wherein X is O, CH₂, S or NH; R¹ is lower alkyl or alkene; lower alcohol(C1 to C22), saturated or unsaturated; or —CH₂CH═CH—(CH₂)₃—COR″ whereinR″ is OH, O— lower alkyl or alkene; R² is OH, NH₂, SH, OPO₃H, loweralkyl or alkene or O— lower alkyl or alkene; and R³ is lower alkyl oralkene or —CH₂—CH═CH—(CH₂)₄—R′″ wherein R′″ is CH₃, CH₂OH, CH₂ —O— loweralkyl or alkene, phenyl or substituted phenyl or

wherein X, R¹, R² and R³ are as defined for formula I and R⁴ is loweralkyl or alkene; lower alcohol (C1 to C22), saturated or unsaturated; or—CH═CH—CH₂—CH═CH—(CH₂)₃—COR″ wherein R″=OH or O— lower alkyl or alkene,or a derivative thereof.
 30. A composition for application to the eyecomprising as active ingredient a compound selected from the groupconsisting of: (a)8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoic acid or aderivative thereof; (b)8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoic acid or aderivative thereof; (c)10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof; and (d)10(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof.
 31. A pharmaceutical composition comprising acompound of the formula:

wherein X is O, CH₂, S or NH; R¹ is lower alkyl or alkene; lower alcohol(C1 to C22), saturated or unsaturated; or —CH₂CH═CH—(CH₂)₃—COR″ whereinR″ is OH, O— lower alkyl or alkene; R² is OH, NH₂, SH, OPO₃H, loweralkyl or alkene or O— lower alkyl or alkene; and R³ is lower alkyl oralkene or —CH₂—CH═CH—(CH₂)₄—R′″ wherein R′″ is CH₃, CH₂OH, CH₂ —O— loweralkyl or alkene, phenyl or substituted phenyl or

wherein X, R¹, R² and R³ are as defined for formula I and R⁴ is loweralkyl or alkene; lower alcohol (C1 to C22), saturated or unsaturated; or—CH═CH—CH₂—CH═CH—(CH₂)₃—COR″ wherein R″=OH or O— lower alkyl or alkene,or a derivative thereof, and a pharmaceutically acceptable carrier. 32.A pharmaceutical composition comprising a compound selected from thegroup consisting of: (a)8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoic acid or aderivative thereof; (b)8(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,10E,14Z-trienoic acid or aderivative thereof; (c)10(S)-hyhydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof; and (d)10(R)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid or aderivative thereof, and a pharmaceutically acceptable carrier.